Today, for lack of appropriate technology there is scant information on how multiple brain areas coordinate their collective neural circuit dynamics. Hence, the recent NIH BRAIN Initiative report calls for new techniques that can provide ?multi-area population recording at cellular resolution?. To address this challenge, we built a large-scale two-photon fluorescence microscope with 16 laser beams that together scan a 4 mm2 area of brain tissue in an awake behaving rodent. This new instrument is the world's largest two-photon microscope and enables Ca2+ imaging studies of the simultaneous dynamics of thousands of individual neurons lying across ~3?7 cortical brain areas. The goal of this High Impact Neuroscience Research Resource Grant is to make this groundbreaking new instrumentation a user-friendly and openly accessible resource for neuroscience researchers around the nation. To achieve this, we will first implement key upgrades to the large-scale microscope that will: improve the usability of the software interface; enable multi-color imaging, including simultaneous dual-color imaging for concurrent Ca2+ imaging of two distinct cell populations; and speed the laser-scanning system to 20 Hz imaging frame rates, to meet the needs of users whose research questions about large-scale neural Ca2+ dynamics require this temporal resolution. We will establish core infrastructure, including a data center dedicated to the computational analysis and bioinformatics needs of the Resource users, and a stock of the transgenic reporter mice and viral vectors that neuroscientists most commonly use to express genetically encoded Ca2+ indicators. We will also hire a full-time staff scientist, who will conduct regular tutorial workshops and directly support individual users regarding training and usage of the instrumentation, adaptation of users' animal behavioral assays for compatibility with large-scale two-photon brain imaging, and data analysis. We plan a staged rollout of service, starting with 5 carefully chosen Phase 1 labs to serve as beta- testers, followed by a larger set of 16 Phase 2 labs, soon followed by opening the R24 Resource to all neuroscience research applicants. To oversee and benchmark progress against quantitative milestones, we have established a Steering Committee of senior scientists, from within and outside our home university, who have complementary areas of expertise ? including in the management of research imaging facilities and public databases of brain imaging data that are broadly shared across the scientific community. The Steering Committee will monitor Resource usage, evaluate user feedback, provide guidance toward superior usability, performance and data sharing, and track the research progress of each neuroscience lab that is using the R24 Resource toward publication of the biological results. The Resource personnel and Steering Committee will also coordinate with the Stanford Neuroscience Institute to provide broad, equitable, well-advertised, and flexible access to the Resource across the local and national NINDS and neuroscience research communities.